A polynucleotide probe comprising the gene encoding a major structural subunit protein of coli surface antigen 6 (CS6) of enterotoxigenic Escherichia coli (ETEC) was developed. Eighty-nine ETEC isolates were examined in parallel with the probe in a colony hybridization assay and in a recently developed polyclonal-antibody-based inhibition enzyme-linked immunosorbent assay (ELISA). The two assays showed a high level of concordance in the detection of CS6-positive ETEC (kappa = 0.84, P < 0.00001). Thus, 36 of the 89 ETEC isolates were identified as CS6-positive by both assays. Six strains that were negative for other colonization factor antigens were positive with the CS6 probe but negative in the ELISA, suggesting lack of surface CS6 expression in these strains. One strain was probe negative but positive in the ELISA, while the remaining 46 strains were negative in both assays. The phenotypic and genotypic assays will prove useful in vaccine-oriented studies of ETEC disease.